Project Summary Cancer genome sequencing has revealed high rates of mutations in chromatin-modifying proteins, highlighting the importance of chromatin regulation in cancer. However, mechanistic links between chromatin and the signaling pathways that are known to promote cancer development remain poorly understood. In melanoma, in addition to other cancers, activation of the MAPK signaling pathway is a driver of tumorigenesis. Current therapeutic strategies used to treat melanoma involve reducing the output of MAPK signaling through pharmacologic inhibition of upstream protein kinases. Unfortunately, resistance rapidly occurs to these therapies, highlighting the need for a more in-depth understanding of downstream signaling. There is an emerging appreciation that chromatin may be a key target for post-translational modifications downstream of MAPK signaling. Preliminary data from the Allis lab and others has established a novel understanding of the role of mitogen and stress activated protein kinases (MSKs) as critical intermediates in MAPK signaling that regulate histone H3 Ser28 phosphorylation (H3S28ph). H3S28ph has a potent role in signal-induced activation of transcription, potentially through the recruitment of p300 acetyltransferase and displacement of Polycomb complexes. MSKs also phosphorylate and activate transcription factors, including NF-?B and CREB, both of which have established roles in melanoma. The ability to dually regulate transcription factors and chromatin puts MSKs in a position to have a major impact on transcription in the context of MAPK pathway activation. Here we will specifically address the hypothesis that constitutive activation of MSKs in melanoma and other cancers controls the transcription of genes that are critical for the growth and maintenance of the tumor. Two Specific Aims are proposed to test this working hypothesis: 1) use a CRISPR-Cas9 system and complementary pharmacologic approaches to determine the requirements of MSK1 and MSK2 for oncogenic transcription in melanoma and 2) use biochemical assays in order to identify new MSK substrates/interacting proteins and understand the mechanisms by which MSKs regulate chromatin and transcription. Importantly, insights resulting from the completion of these aims will potentially lead to novel opportunities for therapeutic intervention in this deadly disease. In addition, BRAF-driven melanoma will be used here as a model cancer type to study the role of MSKs, but MAPK signaling is often dysregulated in cancer, and these findings will serve as the foundation to study these signaling paradigms in various tumor types, such as pancreatic, colorectal, and lung cancer.